Anticonvulsant Activity of Extracts of Plectranthus barbatus Leaves in Mice

Plectranthus barbatus is a medicinal plant used to treat a wide range of disorders including seizure. However, the anticonvulsant activity of this plant has not been studied in depth. We therefore sought to evaluate the anticonvulsant activity of a hydroalcoholic extract of P. barbatus leaves on seizures induced by strychnine sulphate (2.0 mg/kg) and pilocarpine (600 mg/kg) in mice. The extract was administered orally at 1, 10, 30, and 100 mg/kg. We report that the P. barbatus extract had marked anticonvulsant activity against strychnine-induced convulsions, but was quite ineffective against pilocarpine-induced convulsions. Further experiments will be required to identify the active molecules(s) and their mechanism(s) of action.

P. barbatus is also used for the treatment of epilepsy and convulsions [10]; however, only limited data are available concerning the anticonvulsant activity of this plant. The present work was undertaken to evaluate the anticonvulsant activity of an extract of Plectranthus barbatus leaves. Ethical procedures were based on the Brazilian law 6638 (May 8, 1979) "Normas para Prática Didático-Científica da Vivissecção de Animais" and "Ethical Principles for

Evaluation of Anticonvulsant Activity.
For each seizureinducing drug tested, 40 mice were divided into five groups of eight animals; P. barbatus extract was administered by oral gavage at doses of 0, 1, 10, 30, and 100 mg/kg. Thirty minutes after administration, each mouse was injected intraperitoneally with strychnine (2.0 mg/kg) or with pilocarpine (600 mg/kg). The doses of used drugs were defined in a pilot study. The latency to first convulsion and the latency and percentage mortality were recorded for a period of 30 min. Animals surviving more than 30 min were considered to be protected.

Statistical Analysis.
Results are expressed as means ± standard error of the mean (SEM). Comparisons between the averages of series of values were performed by ANOVA followed by Dunnett's multiple comparisons test. Data analysis employed Graphpad INSTAT version 2.0 software; statistical significance was set at P < .05.

Results
P. barbatus extract at doses of 1 and 100 mg/kg significantly (P < .05) delayed the onset of strychnine-induced seizures (Table 1). At a dose of 1 mg/kg, there was a significant (P < .05) increase in the latency to death, but the extract failed to protect the animals against drug-induced death. At a dose of 10 mg/kg, there was no significant difference in time before onset of seizure or death latency. However, P. barbatus extract at dose of 30 mg/kg protected 12.5% (1/8) of treated animals; although time to seizure onset and latency to death were increased by extract administration the difference failed to achieve statistical significance (P > .05). At 100 mg/kg, all treated animals (8/8) were now protected against druginduced death (P < .0001).
For pilocarpine-induced seizures, all P. barbatus extract doses (1, 10, 30, and 100 mg/kg) significantly (P < .05) delayed seizure onset, and doses of 1, 10, and 100 mg/kg significantly (P < .05) increased the latency to death. However, no significant retardation of death latency at a dose of 30 mg/kg was seen compared to the control group, and no dose protected animals from convulsions or death (Table 2).

Discussion
We report that a hydroalcoholic extract of leaves of Plectranthus barbatus exerts anticonvulsant effects against both strychnine-and pilocarpine-induced seizures. All tested doses of P. barbatus extract retarded pilocarpine-induced seizures but failed to protect against death. By contrast, no seizure activity and no death were observed in strychninetreated animals pretreated with the highest tested dose of P. barbatus extract (100 mg/kg).
The mechanism underlying strychnine-induced seizures is thought to involve direct antagonism of strychninesensitive glycine receptors not only in higher brain areas but also in the spinal cord and brainstem, thereby abrogating spinal reflexes and causing motor disturbance, increased muscle tone, hyperactivity of sensory, visual and acoustic perception, tonic convulsions, and death through respiratory or spinal paralysis or by cardiac arrest [11]. Our results demonstrate that strychnine-induced seizures are partly suppressed by P. barbatus treatment.
Pilocarpine is a nonspecific muscarinic acetylcholine receptor agonist; it has been suggested that cerebral structures with a high density of muscarinic receptors are likely to represent the sites of origin of acute pilocarpine seizures [12]. Pilocarpine administration to rodents leads to repetitive limbic seizures and status epilepticus, replicating several features of human temporal lobe epilepsy [13]. It was previously suggested that extracts of P. amboinicus might Evidence-Based Complementary and Alternative Medicine 3 antagonize cholinergic action at muscarinic receptors [14]. On the other hand, P. barbatus extracts were previously reported to inhibit acetylcholinesterase activity [15]. In the present work, the administration of P. barbatus extracts failed to inhibit pilocarpine-induced seizures, even though it retarded the onset of the seizures. Thus, it appears likely that P. barbatus extract presented slight antagonist action in the muscarinic acetylcholine receptors.
Phytochemical studies on P. barbatus have revealed numerous bioactive compounds: notably diterpenoids including forskolin [16], plectranthone J, plectrin, (16S)-coleon E, coleon F, (16R)-plectrinone A, plectrinone B [17], cyclobarbatusin, barbatusin, and 7β-acetyl-12-desacetoxicyclobarbatusin [18]. Of these, forskolin is a promising candidate for the anticonvulsant activity of P. barbatus because this molecule was shown to suppress seizures induced by pentylenetetrazol [19]. Forskolin activates adenylate cyclase and increases intracellular cAMP levels. It was previously reported that forskolin can elicit membrane depolarization in medium spiny neurons of the mouse nucleus accumbens; this was mediated by cAMP stimulation of inward rectifier K + currents [20]. Forskolin increases the amplitude of glycinergic miniature inhibitory postsynaptic currents in the presence of low concentrations of extracellular glycine in isolated rat substantia gelatinosa neurons [21].
In addition to direct effects on ion channels, forskolin treatment is likely to exert neuroprotective effects by increasing cellular levels of neurotrophin-3 (NT-3) [22], a member of the neurotrophin family of neurotrophic factors that support cell survival, axonal growth, and neuronal plasticity [23,24]. Brain injury decreases NT-3 synthesis in the hippocampus [25], whereas NT-3 supplementation can protect neurons against excitotoxic insult [26]. Seizures induced by kainic acid [27] or pilocarpine [28] injection decreased NT-3 expression in the brain of mice, whereas intraventricular infusion of NT-3 in rats inhibited kindling epileptogenesis, mossy fiber sprouting, and increases in hilar area [29], arguing that NT-3 upregulation by forskolin is likely to inhibit seizure development and seizure-related synaptic reorganization.

Conclusion
In conclusion, P. barbatus extract was found to have marked anticonvulsant activity against strychnine-induced convulsions, but was quite ineffective against pilocarpine-induced convulsions. Although forskolin is a promising candidate molecule for the anticonvulsant activity of P. barbatus extracts, it is possible that the bioactivity is mediated by a combination of two or more molecules. Further experiments will be required to identify the active molecules(s) and their mechanism(s) of action.